Characterisation of the signal transduction cascade caused by propofol in rat neurons: from the GABA(A) receptor to the cytoskeleton.

The anaesthetic propofol interacts with the GABA(A) receptor, but its cellular signalling pathways are not fully understood. Propofol causes reorganisation of the actin cytoskeleton into ring structures in neurons. Is this reorganisation a specific effect of propofol as apposed to GABA, and which cellular pathways are involved? We used fluorescence-marked actin in cultured rat neurons to evaluate the percentage of actin rings caused by propofol or GABA in combination with rho, rho kinase (ROK), PI3-kinase or tyrosine kinase inhibitors, with or without the presence of extracellular calcium. Confocal microscopy was performed on propofol-stimulated cells and changes in actin between cellular compartments were studied with Western blot. Propofol (3 microg x ml-1), but not GABA (5 microM), caused transcellular actin ring formation, that was dependent on influx of extracellular calcium and blocked by rho, ROK, PI3-kinase or tyrosine kinase inhibitors. Propofol uses rho/ROK to translocate actin from the cytoskeleton to the membrane and its actin ring formation is dependent on an interaction site close to the GABA site on the GABA(A) receptor. GABA does not cause actin rings, implying that this is a specific effect of propofol.

Association between low concentrations of antibodies to protein alpha and Rib and invasive neonatal group B streptococcal infection.

BACKGROUND: Infection with group B streptococci (GBS) is a serious neonatal disease. The GBS cell surface proteins alpha and Rib elicit protective immunity in animal models and have been suggested as potential antigens in a vaccine against human GBS disease. AIMS: To test the hypothesis that transplacentally transferred maternal antibodies to GBS proteins contribute to the protection of the neonate from GBS infection. METHODS: Thirty neonates with invasive infection were included in a case-control study. IgG antibody concentrations were measured in sera from these neonates, their mothers, and from 60 non-infected controls, neonates as well as mothers. RESULTS: A clear association was found between concentrations of antibody to proteins alpha and Rib in neonatal and maternal sera, indicating that transplacental transfer had occurred. Moreover, low concentrations of antibodies to alpha and Rib in neonatal sera were associated with invasive GBS infection caused by strains expressing the Rib protein. The odds ratio was 0.0007 (95% confidence interval 0.000 to 0.54) for antibodies to alpha and 0.002 (95% confidence interval 0.000 to 0.57) for antibodies to Rib. CONCLUSION: These findings support the notion that antibodies to GBS surface proteins contribute to the protection against neonatal infection.

Appropriate prophylaxis with restrictive palivizumab regimen in preterm children in Sweden.

AIM: Palivizumab (Synagis) was registered in Sweden in 1999 for prophylaxis against respiratory syncytial virus (RSV) in premature infants. The high costs and the limited knowledge of the efficacy of this substance have led to debate about how and when it should be used. National guidelines for the use of palivizumab in Sweden were constructed in the year 2000. The aim of this study was to evaluate the guidelines. METHODS: A nation-wide prospective study was conducted during the two RSV seasons of the years 2000-2002. The paediatric departments in Sweden reported the use of palivizumab, the indication for its use, and the number of infants born preterm before 36 wk of gestation and less than 2 y old who were admitted to hospital for RSV infection. RESULTS: During the two seasons, 218 (3.8%) children who were born before 36 wk of gestation, and 97 (5.4%) who were born before 33 wk, were hospitalized because of RSV infection. Five children were treated with mechanical ventilation. No death caused by RSV was reported. A total of 390 children were treated with palivizumab, and 16 (4.1%) of those who received prophylactic treatment were admitted to hospital with RSV infection. CONCLUSION: We consider the comparatively restrictive Swedish recommendations to be safe and recommend that palivizumab should also be used very restrictively in the future. In our opinion, palivizumab in preterm children could be recommended only for those with chronic lung disease younger than 1 y of age, and with active treatment for their disease.

Localization of the insulin receptor in caveolae of adipocyte plasma membrane.

The insulin receptor is a transmembrane protein of the plasma membrane, where it recognizes extracellular insulin and transmits signals into the cellular signaling network. We report that insulin receptors are localized and signal in caveolae microdomains of adipocyte plasma membrane. Immunogold electron microscopy and immunofluorescence microscopy show that insulin receptors are restricted to caveolae and are colocalized with caveolin over the plasma membrane. Insulin receptor was enriched in a caveolae-enriched fraction of plasma membrane. By extraction with beta-cyclodextrin or destruction with cholesterol oxidase, cholesterol reduction attenuated insulin receptor signaling to protein phosphorylation or glucose transport. Insulin signaling was regained by spontaneous recovery or by exogenous replenishment of cholesterol. beta-Cyclodextrin treatment caused a nearly complete annihilation of caveolae invaginations as examined by electron microscopy. This suggests that the receptor is dependent on the caveolae environment for signaling. Insulin stimulation of cells prior to isolation of caveolae or insulin stimulation of the isolated caveolae fraction increased tyrosine phosphorylation of the insulin receptor in caveolae, demonstrating that insulin receptors in caveolae are functional. Our results indicate that insulin receptors are localized to caveolae in the plasma membrane of adipocytes, are signaling in caveolae, and are dependent on caveolae for signaling.

Respiratory distress syndrome in infants with impaired intrauterine growth.

The recently introduced intrauterine growth curve, based on ultrasonically estimated foetal weights, was retrospectively applied to an inborn population of 883 infants born before 33 gestational weeks at the University Hospital of Lund, during 1985-94. The estimation of birthweight deviation resulted in 630 (71.3%) infants with a birthweight appropriate for gestational age (AGA), 244 (27.6%) infants with a birthweight small for gestational age (SGA) and 9 (1.1%) infants with a birthweight large for gestational age. Birthweight deviation was associated with an increased mortality [odds ratio (OR) adjusted for gestational age 1.29 per SD (12%) change in birthweight for gestational age, 95% CI: 1.10-1.50; p = 0.002]. At gestational age 25-28 weeks, SGA-infants had an increased incidence of respiratory distress syndrome (RDS) as compared to AGA-infants (OR adjusted for gestational age: 1.98, 95% CI: 1.12-3.52; p = 0.019). At gestational age 29-32 weeks, SGA-infants had a lower incidence of RDS as compared to AGA-infants (OR adjusted for gestational age: OR 0.52, 95% CI: 0.34-0.80; p = 0.003). After adjustment for confounding variables, infants born at gestational age 25-28 weeks from mothers with pre-eclampsia, appeared to be a high-risk group for RDS, whereas at the age of 29-32 gestational weeks, negative birthweight deviation had a protective effect against RDS. Antenatal corticosteroid administration appeared to have a less beneficial effect on mortality, RDS and cerebral haemorrhage in infants born SGA vs in those born AGA.

Changing trend in perinatal management and outcome of extremely low birthweight (ELBW) infants.

Perinatal care of the extremely preterm and low birthweight (ELBW) infant is founded on basic principles of physiology and knowledge about the prevailing pathophysiological mechanisms. New therapies in clinical care are usually introduced non-uniformly, so more often there is a gradual rather than a sudden change in the development of perinatal care, conceivably involving also an important learning process. This was confirmed in an evaluation of respiratory care for ELBW infants (n = 325) over a 9-year period (1986-1994). Although birthweight (mean 815 g) and degree of immaturity at birth (mean 26.7 weeks of gestation) did not change over the years, our trend analysis showed that the survival rate increased from 47% to 70% (p < 0.04) and the percentage of survivors without bronchopulmonary dysplasia and/or major intracranial haemorrhages (ICH grades 3 and 4) increased from 67% to 87% (p < 0.006). We suggest that besides medical treatment per se, refinement and tuning of nursing and medical care procedures will also affect the total outcome of ELBW infants.

Low risk of seizure recurrence after early withdrawal of antiepileptic treatment in the neonatal period.

The risk of seizure recurrence within the first year of life was evaluated in infants with neonatal seizures diagnosed with a combination of clinical signs, amplitude-integrated electroencephalogram (EEG) monitoring, and standard EEG. Fifty eight of 283 (4.5%) neonates in tertiary level neonatal intensive care had seizures. The mortality in the infants with neonatal seizures was 36.2%. In 31 surviving infants antiepileptic treatment was discontinued after one to 65 days (median 4.5 days). Three infants received no antiepileptic treatment, two continued with prophylactic antiepileptic treatment. Seizure recurrence was present in only three cases (8.3%)--one infant receiving prophylaxis, one treated for 65 days, and in one infant treated for six days. Owing to the small number of infants with seizure recurrence, no clinical features could be specifically related to an increased risk of subsequent seizures. When administering antiepileptic treatment, one aim was to abolish both clinical and electrographical seizures. Another goal was to minimise the duration of treatment and to keep the treatment as short as possible. It is suggested that treating neonatal seizures in this way may not only reduce the risk of subsequent seizure recurrence, but may also minimise unnecessary non-specific prophylactic treatment for epilepsy.

Pulmonary complications of umbilical venous catheters.

Umbilical venous catheters that are pushed in too far will usually cross the atrial septum and may dislodge in a pulmonary vein. The authors report eight neonates with radiographic documentation of this catheter malposition together with either a localized pulmonary opacification or an autopsy report of a pulmonary hemorrhage or infarction corresponding to the catheter position. The lung injury ranged from a transient edema to hemorrhagic pulmonary infarction and hydrothorax. In two cases autopsy demonstrated localized infarction without previous radiographic signs of catheter-related injury.

Propofol induces changes in the cytosolic free calcium concentration and the cytoskeletal organization of cultured human glial cells and primary embryonic rat brain cells.

BACKGROUND: The site of action of the intravenous anesthetic drug propofol is uncertain. Therefore, we examined the effects of propofol on the cytosolic free calcium levels of cultured primary embryonic rat brain cells (80-85% neurons), and on the organization of the cytoskeleton in these rat cells and in a model system of cultured human glial cells (astrocytes). METHODS: Propofol was added to the cells as the clinically available solution Diprivan. Cytosolic free calcium changes in neurons were studied using Fura2 and a single-cell microfluorometric method. Fluorescence microscopy was used to study the organization of actin filaments and tubulin in detergent-extracted cells. RESULTS: An increase in the cytosolic free calcium concentration of 116 +/- 39 nM was seen shortly after the addition of 0.3 microgram.ml-1 propofol, and a propofol concentration of 0.03 microgram.ml-1 resulted in an increase in cytosolic free calcium concentration of the same magnitude, 119 +/- 42 nM. Most of the calcium (60-75%) came from the extracellular environment, and the rest was from intracellular stores. When neurons were depleted of intracellular calcium by 1,2-bis-5-methyl-amino-phenoxylethane-N,N'-tetra-acetoxymethyla cetate (MAPT/AM), no changes were seen in the actin organization of the cytoskeleton. Actin organization was affected by all concentrations of propofol, 0.3-50 micrograms.ml-1 (1.7-280 microM), when exposure to the drug was achieved by a 30-min incubation. After the incubation, the exposed cells were more rounded and exhibited increased ruffling activity, both at the periphery and on the cellular surface, and ring-shaped actin structures were also seen. These effects were concentration dependent and reversible, and reached a maximum after 20 min of incubation. Propofol had no apparent effect on the organization of tubulin. CONCLUSIONS: Propofol induced changes in the cytoskeletal organization of actin in cultured rat neurons and human glial cells. These changes must have been due to the increase in intracellular calcium seen shortly after the addition of propofol, since no effects on actin organization were seen when intracellular calcium was depleted.

PDGF and neomycin induce similar changes in the actin cytoskeleton in human fibroblasts.

The addition of platelet-derived growth factor (PDGF) to serum-starved fibroblasts induces increased motility, formation of lamellipodia, increased ruffling activity, and actin ring structures associated with dorsal ruffles. Involvement of the phosphatidylinositol cycle (PI-cycle) in these morphological changes was investigated by observing the effects of neomycin, an inhibitor of the PI-cycle, on cultured human foreskin fibroblasts. The role of actin in the changes was investigated by using cytochalasin D (CD). Actin in detergent-extracted cells was labelled with TRITC-phalloidin and examined with fluorescence microscopy. Using PDGF and neomycin simultaneously potentiated lamellipodia formation, ruffling activity, as well as the number of cells with actin rings. Furthermore, neomycin by itself induced morphological changes similar to those induced by PDGF. Quantitation of actin rings showed dose and time dependency for PDGF and neomycin respectively, with a maximal number of cells containing rings after 15 min of exposure to either 3.5 mM neomycin or 10 ng PDGF/ml. Comparing the two substances, PDGF induced ring formation in a greater number of cells. These processes were inhibited by the presence of CD. PDGF- and neomycin-induced changes in the actin cytoskeleton were also observed in human embryonic lung fibroblasts, human glial cells, and embryonic mouse fibroblasts, all of which are known to express PDGF-receptors. In conclusion, the present study indicates that an increased turnover of the PI-cycle is not essential for the changes in actin organization induced by PDGF.

Preservation and visualization of molecular structure in detergent-extracted whole mounts of cultured cells.

Today's electron microscopes have a resolution sufficient to resolve supramolecular structures. However, the methods used to prepare biological samples for electron microscopy often limit our ability to achieve the resolution that is theoretically possible. We use whole mounts of detergent-extracted cells grown on Formvar-coated gold grids as a model system to evaluate various steps in the preparation of biological samples for high resolution scanning electron microscopy (SEM). Factors that are important in determining the structure and composition of detergent-extracted cells include the nature of the detergent and the composition of the extraction vehicle. Chelation of calcium is extremely important to stabilize and preserve the cytoskeletal filaments. We have also demonstrated both morphologically and by gel electrophoresis that treatment of cells with bifunctional protein crosslinkers before or during extraction with detergent can significantly enhance the preservation of both proteins and supramolecular structures. The methods used to dry samples are a major determinant of the quality of structural preservation. For cytoskeletons freeze-drying (FD) is superior to critical point-drying (CPD), one reason being that CPD samples have to be dehydrated, thereby causing more shrinkage as compared to FD samples. The high pressures to which samples are exposed during CPD may also cause increased shrinkage, and water contamination during CPD causes severe structural damage. We have obtained the best structural preservation of detergent-extracted and fixed cells by manually plunging them into liquid propane and drying over night in a freeze-dryer. The factor that most limits achievement of high resolution in SEM is the metal coat, which has to be very thin, uniform, and free of grain in order not to hide structures or to create artifactual ones. We have found that sputter-coating with 1-3 nm of tungsten (W) or niobium (Nb) gives extremely fine-grained films as well as satisfactory emission of secondary electrons. These samples can also be examined at high resolution by transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). The best preservation and visualization of supramolecular structures have been obtained using cryosputtering, in which the samples are freeze-dried and then sputter-coated within the freeze-dryer while still frozen.

Cryosputtering--a combined freeze-drying and sputtering method for high-resolution electron microscopy.

Preparing cellular structures for visualization by high-resolution scanning electron microscopy (SEM) is a multi-step process which includes fixation, dehydration, drying and metal coating. Drying and metal coating are limiting for high-resolution work. Commonly, the dried samples are exposed to the air before they are inserted into a metal coating apparatus, thereby exposing them to moisture and the accompanying risk of rehydration, which may cause changes in the supramolecular structure. We have modified a freeze-dryer to accommodate a magnetron sputtering head, in order to sputter-coat the frozen-dried samples while still in the drying chamber in the cold, a process we call cryosputtering. A layer of 1.5 nm of tungsten was cryosputtered onto whole mounts of cytoskeletons from detergent-extracted human glioma cells or fibroblasts and the specimens were examined by high-resolution SEM and transmission electron microscopy (TEM). To reduce the effects of backstreaming oil from the vacuum system, a turbomolecular pump backed by a two-stage rotary vane pump was connected to the drying-coating chamber. This pump system provides a high vacuum, making it possible to dry the specimens at -90 degrees C/183 K, thus reducing the risk for recrystallization of water. Furthermore, the high vacuum minimizes the negative effects of contaminants, which can be deposited onto the specimen surface and affect the quality of the metal coat formed during sputtering.

Spontaneous pneumopericardium in an otherwise healthy full-term newborn.

Neonatal pneumopericardium is usually a complication of mechanical ventilation in premature infants with respiratory distress syndrome. We report a full-term neonate who developed pneumopericardium after forceps delivery and mild asphyxia. The child was never ventilated and had no signs of parenchymal lung disease. The pneumopericardium resolved spontaneously. Although drainage of pneumopericardium is usually recommended, this may not always be necessary when there are no signs of cardiac tamponade.